The proposed research will continue in directions already well advanced during the six years of this grant and move into new directions: (a) Mercury resistance and resistance to other toxic heavy metals will be studied, starting with the determination of the plasmid (R-factor) genetic basis of the resistance, progressing through the physiological basis, (which in the case of mercury is the reduction of Hg(II) to metallic Hg(O) and volatilization of metallic Hg(O) from the medium), followed by isolation and purification of the enzymes involved to homogeneity. After studying the mechanism of the reaction (cofactors, substrates, products) with purified enzymes, mutants defective in the enzyme and regulatory mutants will be isolated and the process repeated. (b) Oxidative phosphorylation in Escherichia coli and the related processes of active transport across the membrane and elctron transport will be studied by a similar approach to that above. Physiological experiments, defining the systems and interrelations, followed by isolation and mapping of mutants defective in various aspects of cellular bioenergetics, followed by isolation and purification of the macromolecules (proteins) determined by the genes will be the plan of attack on this complex problem. Cation transport systems extensively studied in this lab will be used in these studies as will colicins that interfere with cellular energy metabolism. This laboratory has had some degree of success in such an approach during the last six years in particular in our studies of colicin action, cation transport and mercury resistance. These will be continued with a more biochemical approach and the new direction of studies of energy metabolism, in particular oxidative phosphorylation, will be emphasized.